The monoamine oxidases (MAO, EC 1.4.3.4) are anzymes responsible for the oxidation of biogenic amines. Due to their important role in the neurotransmitters inactivation, the dysfunction of MAO enzymes (increased levels of MAO activity) is associated with a number of mental and neurological disorders such as depression, anxiety disorders and migraine.
The MAO enzymes exist in two isoforms, MAO-A and MAO-B, which have approximately 70% amino acid sequence identity (Oesch and Arand, Toxicology, Academic Press, San Diego USA, 1999) and differing in substrate specificity and tissue distribution (Bach et al., Proc. Natl. Acad. Sci. USA 85, 4934-4938, 1988). The MAO-B enzyme is found in high levels in the liver, platelets, and especially in brain (Cesura and Pletscher, Prog. Drug Res. 38, 171-297, 1992). The natural substrates for MAO-B are preferentially phenylethylamine and tyramine. Another important substrate for MAO-B is the tertiary amine 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which is metabolized by MAO-B in neurons. It is known, that the product of this metabolic reaction MPP+ is a Parkinson inducing dopaminergic neurotoxin (Youdim et al., Biochem. Pharmacol. 41, 155-162, 1991; Petzer et al., Bioorg. Med. Chem. 11, 1299-1310, 2003).
It is known, that MAO inhibitors (MAOI) are substances that inhibit the MAO activity and due to their selectivity of the MAO receptor, they can be selective or non-selective. MAO-A inhibitors are therapeutically useful as antidepressants, whereas MAO-B inhibitors can be used in the monotherapy or in combination with levodopa (L-DOPA) for the treatment of Alzheimer's Disease (AD), Parkinson's Disease (PD) and other neurological diseases associated with the degeneration of dopaminergic neurons (Plhagen et al., Neurology, 66, 1200-1206, 2006). The World Health Organisation (WHO) reported that 36.5 million people worldwide are living with different types of dementia including Alzheimer's Disease and Parkinson's Disease Dementia (PDD)—with upward tendency (WHO, Dementia: a public health priority, 2012). These neurodegenerative diseases are the most common types of dementia, characterized by a decreased level of dopamine which is mediated by the degeneration of dopaminergic neurons in substantia nigra.
The current therapy of AD and PD is primarily focused on the treatment of the symptoms affecting the quality of live in patients. Since these symptoms are caused by a decreasing of dopamine levels in the brain, the most therapeutic drugs are based on dopamine replacement using dopamine-enhancing approach such as levodopa or dopamine agonists to stimulate the dopamine receptors. The MAO-B inhibitors are also used as an alternative therapeutic approach of the dopamine agonists for treatment of neurodegenerative diseases. For example, both selegiline (Riederer and Lachenmayer, J. Neural Transm., 110 (11), 1273-8, 2003) and rasagiline (Lakhan, Molecular Neurodegeneration, 2(13), 2007) are used as irreversible inhibitors for the treatment of PD. The therapeutic effect of MAO-B inhibitors is provided by blocking of monoamine oxidase B (MAO-B) enzymes in the brain.
Patent application EP1403255 discloses a number of substituted 1H-indazole derivatives, including 1H-indazolecarboxamide and 1H-indazolemethyleneamine. These compounds and prodrugs thereof are described as Rho kinase inhibitors (ROCK-II inhibitor, ROC alpha inhibitor) and are useful as prophylactic or therapeutic agents for urinary incontinence.
It is therefore an object of the present invention to provide in vitro selective and reversible MAO-B inhibitors with IC50 values in the subnanomolar range for the prevention and treatment of acute and chronic neurological disorders, cognitive and neurodegenerative diseases.
Description
The present invention relates to the use of substituted indazole derivatives represented by the formula I:

wherein L is —CO—NH— or —CH═N—;
A1 is —N, —CH or N—(CH2)nR1;
A2 is —N or —CH;
R1 is a hydrogen atom, or represents branched or unbranched —(C1-C3)-alkyl, —(C1-C3)-alkyl, wherein one, two or three hydrogen atoms may be substituted by a halogen, —(C1-C3)-alkoxy or halogen-(C1-C3)-alkoxy; mono- or dihydroxy-(C1-C3)-alkyl;
R2 and R3 both together or independently from each other are halogen, hydroxy, halogen-(C1-C3)-alkyl, —(C1-C3)-alkoxy, —O—(C1-C3)-alkoxy;
wherein R2 and R3 both together or independently from each other are aryl, aryl substituted by a (C3-C5)-alkyl, when A is —CH;
and wherein R2 and R3 both together or independently from each other are heteroaryl, substituted by a (C3-C4)-alkyl, when A is ═N;
and n is 0, 1, 2 or 3,
their pharmaceutically acceptable salts, isomers or mixtures thereof as in vitro selective and reversible MAO-B inhibitors with IC50 values in subnanomolar range for the manufacture of medicaments for the prevention and treatment of acute and chronic neurological disorders, cognitive and neurodegenerative diseases. In particular, the compounds of formula I are useful for the prevention and treatment of neurodegenerative disorders such as Parkinson's Disease, Alzheimer's Disease and dementia.
In one more preferable embodiment, for the manufacturing of a medicament can be used compounds of formula I selected from the group consisting of:
N-(3,4-Dichlorophenyl)-1H-indazole-5-carboxamide,
N-(3,4-Dichlorophenyl)-1H-indole-5-carboxamide,
N-(3-Chloro-4-methoxyphenyl)-1H-indazole-5-carboxamide,
N-(4-Chloro-3-methoxyphenyl)-1H-indazole-5-carboxamide,
N-(3-Chloro-4-hydroxyphenyl)-1H-indazole-5-carboxamide,
N-(4-Chloro-3-hydroxyphenyl)-1H-indazole-5-carboxamide,
N-(3,4-Dimethoxyphenyl)-1H-indazole-5-carboxamide,
N-(3,5-Dichlorophenyl)-1H-indazole-5-carboxamide,
N-(3-Chloro-4-fluorophenyl)-1H-indazole-5-carboxamide,
N-(4-Chloro-3-fluorophenyl)-1H-indazole-5-carboxamide,
N-(3,4-Difluorophenyl)-1H-indazole-5-carboxamide,
N-(5,6-Dichloropyridin-3-yl)-1H-indazole-5-carboxamide,
N-(3,4-Dichlorophenyl)-1-methyl-1H-indazole-5-carboxamide,
N-(3,4-Dichlorophenyl)-2-methyl-2H-indazole-5-carboxamide,
mixture of N-(3,4-Dichlorophenyl)-1-methyl-1H-indazole-5-carboxamide and
N-(3,4-Dichlorophenyl)-2-methyl-2H-indazole-5-carboxamide,
N-(3,4-Dichlorophenyl)-1-methyl-1H-indole-5-carboxamide,
N-(3-Chloro-4-fluorophenyl)-1-methyl-1H-indazole-5-carboxamide,
N-(3-Chloro-4-fluorophenyl)-2-methyl-2H-indazole-5-carboxamide,
N-(3,4-Difluorophenyl)-1-methyl-1H-indazole-5-carboxamide,
N-(3,4-Difluorophenyl)-2-methyl-2H-indazole-5-carboxamide,
N-(3,4-dichlorophenyl)-1-(2-methoxyethyl)-1H-indazole-5-carboxamide,
(E)-N-((1H-indazol-5-yl)methylene)-3,4-dichloroaniline,
(E)-N-((1H-indazol-5-yl)methylene)-4-chloro-3-fluoroaniline,
(E)-N-((1H-indazol-5-yl)methylene)-3-chloro-4-fluoroaniline,
(E)-N-((1H-indazol-5-yl)methylene)-5,6-dichloropyridin-3-amine,
(E)-3,4-dichloro-N-((1-methyl-1H-indazol-5-yl)methylene)aniline
and their pharmaceutically acceptable salts.
The compounds within this invention represented by the general formula I may be used for preparing of radiolabeled [3H]-analogues of the compounds of the present invention, which can be useful as MAO-B radioligands in radioligand binding studies related to MAO-B inhibition.
The compounds of formula I may be prepared by any process known by one skilled in the art. In one preferred embodiment, the compounds of the present invention are prepared following the synthetic methods showed in Scheme 1:

wherein A1, A2, R1, R2, R3 and L have the above meanings and wherein R4 and R5 are carboxylic or substituted carboxylic group or an aldehyde group.
According to method A and C, an amide bond is formed by the reaction of the differently substituted carboxylic acids with general formula IIIa and IIIb with the amino group of the corresponding disubstituted anilines or aminopyridines with general formula IV. The amide coupling reaction can be conducted in the presence of a base such as Hünig's base (N,N-diisopropylethylamine, DIPEA) and a condensation agent such as e.g. O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU), or other benzotriazol derivatives. The reaction can be conducted in suitable solvent such as methanol or more preferably in acetonitrile (ACN) at room temperature over night (method A). Fort the amide bond forming step another condensation reagents like carbodiimides such as e.g. N,N-dicyclohexylcarbodiimide (DCC) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) or other suitable coupling reagents and methods can be used. This step can be conducted at room temperature in the presence of methanol, N,N-dimethylformamide (DMF) or other suitable solvents (method C).
Another method B to prepare compounds of formula I involves the use of activated carboxylic acids, e.g. acid chloride intermediates of formula IIIa. This method is described by Raffa et al. (Arch. Pharm. Chem. Life Sci., 342, 265-273, 2009; II Farmaco 57, 183-187, 2002).
For the preparation of a compound of formula I wherein L has the meaning of —CH═N—, the optionally substituted aldehyde with general formula IIIa can be condensed with an amino group of the aniline or aminopyridine IV. The reaction is carried out in the presence of catalytic amount of acid such as e.g. acetic acid by using suitable solvent such as ethanol at room temperature or preferably under reflux. The reaction takes from several minutes to 24 hours (method D). As described in the international patent application WO2011/076786, the product of this process is only possible on condition that the other amino group containing reagent, e.g. an nitrogen in case of aminopyridine, is stable under the described conditions.
In accordance with the present invention wherein L is —CO—NH— and —CH═N—, A1, A2, R1, R2 and R3 are defined as mentioned above, the N-alkylated compounds of the formula I can be optionally prepared via intermediates thereof, e.g. compounds of general formula Ia, as shown in Scheme 1.
Intermediates of formula Ia wherein R1 is hydrogen and n is 0 can be defined as final compounds. The compounds of formula Ia can be obtained as described above by using method C or D. To introduce an additional substituent in the compounds of general formula I, e.g. an alkyl group in the position 1 or 2, more preferably in the position N1, the intermediates Ia can be alkylated with an appropriate alkylating reagent such as alkylhalide or methyl methanesulfonate (MMS). The reaction occurs in the presence of an excess of a base such as potassium carbonate in suitable solvent such as N,N-dimethylformamide (DMF) at room temperature. Depending on the alkylating reagent, the reaction can be achieved at temperatures between 60° C. and 90° C. for 3 up to 26 hours (alkylation method E). The conversion of the starting material is detected by TLC. The mixture of the N1-/N2-alkyl products with general formula I can be separated by column chromatography on silica gel (eluent: dichloromethane/methanol 9:1).
According to method E, compounds selected from the formula I of the present invention can be radiolabeled by an alkylation reaction with the corresponding radiolabeled alkylating reagent, e.g. methylation with tritium-labeled methyl methanesulfonate [3H]MMS.
The pharmaceutically acceptable salts of the compounds of formula I may be prepared by adding free organic and inorganic acids. Among organic acids, trifluoroacetic acid, acetic acid, oxalic acid, or lysine acid may be used to form pharmaceutically acceptable salts of the compounds represented by the formula I of the present invention. Among inorganic acids, hydrochloric acid, hydroboronic acid, sulphuric acid, or phosphoric acid may be used for the preparation of the pharmaceutically acceptable salts thereof.
The MAO-A and MAO-B enzyme activity of the compounds was estimated following an assay adapted from the method described by Holt et al. (Anal. Biochem., 142, 627-637, 1997). The MAO experiments were performed using a fluorescence-based detection of resorufin, obtained by a reaction of released from the biological sample H2O2 with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in the presence of a peroxidase (horseradish peroxidase-coupled reaction, HRP).
The inhibitory activity of the compounds of general formula I was investigated at rat and human MAO-A and MAO-B using rat and human MAO-A and MAO-B enzymes, respectively.
To perform rat MAO-A and MAO-B experiments, a preliminary membrane preparation of mitochondrial-enriched rat livers isolated from male Sprague-Dawley rats (Harlan Sprague Dawley, Dublin, US) was required. As sources for recombinant human MAO-A and MAO-B enzymes were used microsomal products, prepared from baculovirus-infected insect cells expressing human MAO-A (Sigma-Aldrich, M7316) and human MAO-B (Sigma-Aldrich, M7441), respectively.
To estimate MAO-A and MAO-B inhibitory activity of the compounds of formula I at rat MAO-A and MAO-B as well as at human MAO-A and MAO-B, the corresponding test compound was dissolved in 100% DMSO and subsequently added to the appropriate enzyme solution containing rat liver mitochondria (rat MAO-A and MAO-B) or recombinant human protein (for determination of human MAO-A and MAO-B) in sodium phosphate buffer. The test sample was incubated for 30 min at room temperature prior to the addition of Amplex® Red reagent (Invitrogen A12214) using for the fluorescence-based measuring of MAO inhibitory activity. The enzymatic reaction started by the addition of the substrate p-tyramine. The fluorescence measurements were performed over a period of 45 min using a microplate fluorescence reader (excitation at 544 nm and emission at 590 nm). IC50 values were determined from inhibition curves obtained using different inhibitor concentrations in triplicate within the same experiment, by fitting data to a four parameter logistic equation using computer program.
The time-dependent inhibition studies of the compounds selected from the formula I were deduced at their corresponding IC80 values versus p-tyramine (estimated at 150 μM final concentration) in the test samples with and without enzyme inhibitor. The enzyme activity of the test compounds was measured for (20-30) 22 min in the presence of low substrate concentration of following by a gradually increasing of the substrate concentration of p-tyramine. A reactivation of MAO-B activity was observed after increasing of the substrate concentration. The reactivation of the enzyme was monitored by fluorescence measurements over a period of 5 hours. In the case of the test compounds of formula I, an elevated fluorescence can be detected, indicating that the test compounds are reversible MAO-B inhibitors. The irreversible inhibitor selegiline (Youdim et al., Br. J. Pharmacol., 132, 500-506, 2001) and the reversible inhibitor safinamide (Maj et al., Eur. J. Pharmacol., 359, 27-32, 1998; Marzo et al., Pharmacol. Res., 50, 77-85, 2004) were used as reference compounds.
The substituted indazole derivatives of general formula I related to the present invention and their pharmaceutically acceptable salts, isomers or mixtures thereof can be used as ingredients in the form of pharmaceutical preparations with oral or parental administration. The pharmaceutical preparations can be used in the form of hard or liquid dosage forms, prepared in known manner by means of common excipients and techniques.